Biomaterial/Medical device testing

The biocompatibility testing of materials used in single or multicomponent medical devices for human use depends to a large degree on the nature of the end-use application. Guidelines for evaluating the safety of materials used in medical devices are available from several sources including the FDA Modified ISO Matrix (Blue Book Memorandum # G95-1, Attachment A), USP, Advamed, American Society for Testing and Materials (ASTM Vol. 13.01, Practice F748-83) and AAMI Standards and Recommended Practices (Volume 4).

The medical device development process is multidisciplinary in nature, from the choice of a candidate material, through characterization of the finished product. Testing should take place at several logical points in the development of new materials and devices.

Although the selection and timing of a particular test will differ from one device to another, the evaluation procedure falls into four phases:

1. Preliminary screening of raw materials, i.e. cytotoxicity, hemolysis
2. Biocompatibility tests of device components, per the FDA Modified ISO Matrix
3. Final product evaluation
4. Release and audit testing, e.g., Bacterial Endotoxin Testing
   

Pyrogen Test (USP <151>)
Pyrogenic (fever producing) substances are either endotoxins derived from gram-negative bacteria or are chemical in origin. The purpose of the test is to determine if the material or device is nonpyrogenic. Pyrogen testing should be considered in the evaluation of devices and materials as described in ISO 10993-11.

Bacterial Endotoxin Test (USP 85)
The Limulus amoebocyte lysate (LAL) test is based on the observation that bacterial endotoxins react with a lysate derived from circulating cells associated with the blood clotting mechanism of the horseshoe crab, Limulus polyphemus. The LAL test is recommended for the evaluation of medical devices. Note that the LAL test only detects bacterial endotoxins, not chemical pyrogens. A validation program is required to satisfy FDA requirements for the use of the LAL test.
 

Bacterial Endotoxin Test Validation (USP 85)
Each sample tested by the BET method must first be validated. Three lots of the material or device extract are tested using an inhibition and enhancement curve. If the results of the product eluate spiked with endotoxin mimic the standard curve, the material or device extract is considered validated for use.

Safety Test, General (USP <85>)
This test evaluates systemic toxicity of transfusion and infusion assembly extracts. For other non-biological products, variations of the test dose and route of administration are product specific. Test samples are injected into mice—mice are observed over a 48 hour period for survival and overt toxicity.

Safety Test, Biologics (USP <88>)
The safety test for biologics (or biological products) is part of the test battery required by the Food and Drug Regulations (21 CFR parts 600-680) for lot validation of licensed biological products. At least two mice and two guinea pigs are injected intraperitoneally with a test dose of the product, and the animals are observed over seven days. The test requirements are met if the animals survive the test period, do not exhibit toxic responses, and show no weight loss.

Systemic Injection Test (USP 88/ISO 10993-11)
The acute toxicity test is designed to provide direct assessment of adverse systemic effects. Up to 4 different extracts (saline, 5% alcohol/saline, polyethylene glycol 400 and vegetable oil) of the test material and the corresponding blanks are prepared. Groups of five mice are injected intravenously or intraperitoneally with each test or negative control extract. The mice are observed for three days.
Abnormal Toxicity
Abnormal Toxicity is a European Pharmacopeia standard for assessment of biological products. The test material is administered to mice and guinea pigs, which are then evaluated for signs of toxicity.
 

Intracutaneous Reactivity Test (USP 88/ISO 10993-10)
This acute toxicity test is used to determine the irritant effect of toxic leachables present in extracts of test materials. Up to 4 extracts are prepared (saline, 5% alcohol/saline, polyethylene glycol 400 and vegetable oil) of the test sample and corresponding blanks. For the USP test method, groups of two rabbits are injected subcutaneously with each test extract (ten sites) or the corresponding blank (five sites). The ISO procedure requires 3 animals per extract. Animals are scored daily for three days post-treatment.
 

Implantation Test - 1, 4, 12, 26, 52 Weeks (USP 88/ISO 10993-6)
Intramuscular implantation of test materials in rabbits provides a direct assessment of the acute or long-term tissue response to the toxic effects of leachable substances from candidate biomaterials. The length of the implant period is determined by the end-use of the material. The USP procedure requires that two rabbits be implanted with a minimum of four strips of test material and two strips of negative control plastic via a trocar into the paravertebral muscles. Three rabbits are used for the ISO procedure with 4 test and 4 control implants. Macroscopic evaluation of tissue response to implants of the test material are compared to the corresponding negative control sites. When the test material cannot be molded into the dimensions that would allow for trocar implantation, the test material is implanted using surgical methods. Subcutaneous implantation is also available.

Histology of Implant Sites (ISO 10993-6)
The implant sites are microscopically evaluated by a veterinary pathologist. The tissue sections are scored for inflammation, necrosis, fibrosis and other indicators of a toxic interaction between muscle tissue and test material. An overall toxicity rating of the test material compared to a negative control is reported.

Primary Skin Irritation (ISO 10993-10)
The test material is applied as a patch to intact and/or abraded skin. After a period of time defined by the sponsor, the sites of application are evaluated for irritation.
 

Skin Sensitization, Maximization Method (ISO 10993-10)
The Guinea Pig Maximization Test (GPMT) consists of a two-stage induction procedure over three weeks using intradermal injection of Freund's Complete Adjuvant (FCA) followed by a single application (topical patch) of the test substance or extract to the skin. Fourteen days after the topical phase, the animals are challenged. The test sites are evaluated for sensitization at 24, 48, and 72 hours.

Skin Sensitization, Closed Patch Test (ISO 10993-10)
This method is a repeated topical application (induction and challenge) method using occlusive patches. The test material is applied directly to the skin. After several exposures, the sites are challenged to determine delayed contact sensitization.

Biological Reactivity Tests, in vivo (USP ) {Class Plastics Testing}
These USP tests are designed to evaluate the biological response to plastics for use in medical devices, implants, containers, and base materials. The choice of a particular test battery is dependent on the end use of the test material. The tests include: Systemic Injection; Intracutaneous Reactivity; and Muscle Implantation.

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Acute Ocular Irritation
Lenses exposed to a sponsor specified lens regimen or test solution are inserted/instilled into rabbit eyes. Each rabbit eye is scored up to twice daily using the Draize method to determine swelling, redness or corneal damage. Ophthalmoscope and slit lamp examinations are made before and at the end of a five day study period. 

Acute Ocular Irritation Test (21 Days) (FDA)
This test is designed to determine the ocular irritation and toxicity of contact lenses, associated lens care regimen(s) and solutions over 21 days in rabbits' eyes. Generally, 8 to 12 rabbits with clinically normal eyes are used in the study. The sponsor must specify the lens care regimen to be employed. Rabbits' eyes are examined daily and scored using the Draize system. Before treatment and at 7, 14 and 21 days, the eyes of each rabbit are also examined with an ophthalmoscope and scored for ocular irritation using the McDonald-Shadduck method (slit-lamp and fluorescein stain). At the termination of the study, the eyes are removed for histopathology evaluation and corneal lactic acid metabolism determination, (if appropriate).

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Cytoxicity testing

Mammalian tissue culture systems are currently used to evaluate biocompatibility and toxicity of materials for use in medical devices and associated products. Cell culture testing methods have shown good correlation with animal assays and are frequently more sensitive to toxic materials. Several of these cytotoxicity test procedures are widely accepted in biomaterial screening, quality control and audit programs. In general, these in vitro techniques use a variety of cell types which differ in relative sensitivity and the time required to conduct the assay. Results obtained with cell culture methods must be evaluated in conjunction with supporting or associated in vivo studies and with the end use of the product. 

Direct Contact (ISO 10993-5)
Cell cultures are grown to a standard monolayer. The test material is placed in direct contact with the cell layer for 24 hours. Subsequently, the monolayers are examined microscopically for the presence of morphological changes, reduction in cell density or lysis induced by the test material. A USP procedure is also available. 

Agar Diffusion - Direct Contact or Saline Extract (ISO 10993-5)
The cell monolayer is overlaid with agar and stained before treatment with the test material or extract. After 24 hours, the cells are scored microscopically for decolorization and lysis. A USP procedure is available also. 

MEM Elution - Test on Extracts (ISO 10993-5)
The test material is extracted for 24 hours in Minimum Essential Medium (MEM). An extract is prepared from the test material which is then placed on cell monolayers. The cells are examined for morphologic changes and cytolysis to determine a toxicity score. 

MEM Elution - End Point Titration
Extracts of test materials shown to be toxic in the MEM Elution Test are serially diluted to determine the highest dilution for which there is no toxic response. 

Inhibition of Cell Growth
Extracts or solutions are evaluated for inhibition of cell growth (ICG) by determining the protein content of monolayers at zero time and 72 hours after incubation. This procedure can be used for evaluation of medical device plastics or intra-ocular lenses. 

Virucidal Efficacy Test
Test contact lenses are first inoculated with organic soil and Herpes simplex virus (HSV), then treated with a sponsor specified lens care and disinfection regimen, and finally transferred to VERO cell monolayers for absorption of surviving virus particles. Associated solutions are also neutralized and exposed to VERO monolayers. The VERO monolayers are monitored every two days for a ten day period to determine virus-specific cytopathic effect (CPE) and/or cytotoxicity induced by the test lens or the neutralized cleaning/disinfection solution(s). This test is required by the FDA Guidelines for Class III contact lenses and solutions. Additional viruses/cell lines are available. 

D Value Determination (Disinfectants):
Herpes simplex virus (HSV) suspensions are treated with a sponsor specified-disinfection regimen(s) and aliquots are withdrawn over time to determine the presence or absence of virus-specific cytopathic effect in VERO cell monolayers. The D value is calculated from the determination of the Tissue Culture Infectivity Dose (TCID) 50 of HSV at each time of sampling. The time required to reduce the HSV concentration (infectivity) by 90% is reported as the D value. Other viruses such as Polio and Adenovirus are available for testing. 

Germicide/Sterilant Virucidal Challenge Tests:
The test material is challenged with HSV and/or poliovirus. Several different test methods are available and may be tailored to the specific needs of the client.

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Hemocompatibility testing: (ISO 10993-4)

Hemolysis Test - Direct Contact
The test sample is placed in direct contact with an aliquot of saline containing rabbit red blood cells. The percent hemolysis induced by the biomaterial incompatibility is determined by spectrophotometric measurement of hemoglobin content.

Hemolysis Test — Extract
The test sample is extracted with saline for 30 minutes. A portion of the saline extract is removed and added to a standard aliquot of diluted rabbit blood cells and the two are mixed gently. The percent hemolysis induced by biomaterial leachables is determined by spectrophotometric measurement of hemoglobin release.

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Mutagenicity testing (ISO 10993-4)

There is a recognized relationship between mutagenicity and carcinogenicity of chemical agents, which indicates that an assessment of the potential mutagenicity of leachable components is useful in the evaluation of candidate biomaterials and medical devices.

Ames Mutagenicity Test
The objective of the test procedure in the Ames Salmonella plate assay is to evaluate the test article extract (saline or DMSO) for mutagenic activity in any of five histidine-dependent test strains of Salmonella typhimurium with and without activation with mammalian microsomes (Arochlor induced rat liver S9 fraction). Parallel negative control (extraction media) and positive control (known mutagen) plates are included. In general, test articles which produce a positive dose response over three concentrations, with the highest increase equal to three times the solvent control value, are considered mutagenic.

NOTE: Other mutagenicity tests are available upon request.

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Mucosal irritation testing: (ISO 10993-10)

Primary Ocular Irritation
Saline and vegetable oil extracts are made of the biomaterial. The extracts are topically instilled into one eye of each of six rabbits. The contralateral eye receives the negative control (blank) extract. Test solutions may be instilled directly (after appropriate in vitro screening). Each rabbit eye is scored up to twice daily for three days to determine conjunctival swelling, both redness or corneal damage. Ophthalmoscope and slit lamp examinations are performed before and three days after extract administration.

Vaginal Irritation Test
The test material is inserted into the vagina of each of three rabbits. After sponsor specified time, each rabbit is euthanized. The vaginal tissue is removed and evaluated by a veterinary histopathologist.

Hamster Cheek Pouch
The test material is sutured inside the hamster cheek pouch. After sponsor specified time, each hamster is euthanized and the tissue removed for histopathological evaluation.

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General toxicology studies

The following tests are used to evaluate a variety of chemicals and biomaterials. The specific study design for acute or subchronic toxicity testing is chosen to meet the regulatory requirements and the proposed end use of the material.

Acute toxicity is defined by Organization for Economic Cooperation and Development (OECD) as the adverse effects occurring within a short time of administration of a single dose of a substance or multiple doses given within 24 hours. The objectives of acute toxicity testing are to define the intrinsic toxicity of the chemical, to identify target organs in susceptible species, to select dose levels for subchronic studies, and to provide information for risk assessment after acute exposure.

Subchronic toxicity is defined by OECD as the adverse effects occurring as a result of the repeated daily dosing of chemical to experimental animals for part (not exceeding 10%) of the life span. Properly designed, subchronic studies give information on the cumulative toxicity of a substance, target organs and the physiological and metabolic tolerance of a compound at low dose prolonged exposure. Subchronic implants are available also. 

Acute Dermal Toxicity
Ten guinea pigs are treated with the test material on intact and/or abraded skin for exposure times of either 4 hours (OECD) or 24 hours (EPA). The animals are observed for signs of toxicity and lethality for up to 14 days. 

Acute Oral Toxicity
Ten rats are given an oral dose of the test material by gavage in a constant volume of the appropriate solvent vehicle. Clinical signs of toxicity, morbidity or mortality are observed up to 14 days subsequent to treatment. 

Acute Toxicity (Limit Test) 

Acute Toxicity (Up-Down Bioassay) 

Subacute Toxicity (5 Days) Subchronic Toxicity (90 Days)

Acute, subacute, subchronic, and chronic toxicity testing are performed according to sponsor's specifications.

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Specialized services

Pharmaceutical Evaluations
Tests such as systemic toxicity, acute oral toxicity, pyrogen testing are available.

Antibody Production
Moog is experienced in the production of polyclonal and monoclonal antibodies in rabbits, rats, and mice using client specified protocols. We are familiar with all principal routes for delivery of antigenic material (subcutaneous, intramuscular, intraperitoneal, intratracheal, intravenous, rectal, oral, nasal and aerosol) to experimental animals, as well as blood sampling techniques (tail vein, orbital, ear vein, vena cava, cardiac puncture) and tissue harvest (hybridoma cells, ascites fluid, spleen). Facilities are available for both short and long term antibody production studies. We have experience in handling biohazardous agents at the Biosafety Levels 1 and 2.

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